Deoxyribonucleic acid coding for glutathione-S-transferase and its use

ABSTRACT

The present invention relates to a novel deoxyribonucleic acid (DNA) and its use for the transformation of vectors, host organisms and plants and for the breeding of plants with a high resistance to herbicides.

The present invention relates to a novel deoxyribonucleic acid (DNA) andits use for the transformation of vectors, host organisms and plants andfor producing plants which have increased resistance to herbicides.

The genetic modification of plants so that they have increasedresistance to particular herbicides has already been disclosed. Thismakes it possible to employ herbicides which have low selectivity butotherwise advantageous properties in crops of those plants which, in theoriginal non-transgenic form, would be damaged by these herbicides.Thus, the provision of herbicide-resistant plants increases theselection of herbicides which can be employed, and in many cases it isalso possible to make do with relatively low herbicide applicationrates, for example if control of the unwanted plants can take place onlyafter their emergence when the particular damage threshold is reached.There is thus a considerable need to produce novel crop plants whichhave increased resistance to other herbicides.

Glutathione S-transferases are multifunctional proteins which make aconsiderable contribution to the detoxification of cytotoxic substances.The enzymes catalyze the conjugation of reduced glutathione toelectrophilic hydrophobic substrates which may be of natural orsynthetic origin.

The physiological substrates of glutathione S-transferases in plants andtheir role in plant metabolism are not known in detail. However, it hasbeen demonstrated that these enzymes are involved in the detoxificationand thus in the mechanism of selectivity of a number of importantherbicides from the group of thiocarbamates, chloroacetanilides andS-triacines: Mozer T. J., Tiemeier D. C., Jaworski E. G., Biochemistry22:1068-1072 (1983); Moore R. E., Davies M. S., O'Connell K. M., HardingE. I., Wiegand R. C., Tiemeier D. C., Nucleic Acids Res. 14:7227-7235(1983); Grove G., Zarlengo R. P., Timmermann K. P., Li N., Tam M. F.,Tuc C. P. D., Nucleic Acids Res. 16:425-438 (1988).

The novel deoxyribonucleic acid which codes for the novel proteinglutathione S-transferase IIIc ("GSTIIIc" hereinafter), which has theamino-acid sequence listed in SEQ ID NO: 2, has now been found (thenovel DNA according to the invention being referred to as "GSTIIIc DNA"hereinafter).

It has furthermore been found that plants into whose genome the novelGSTIIIc DNA has been incorporated have an increased resistance, bycomparison with the corresponding "starting plants", to herbicides,preferably heteroaryloxyacetamide herbicides.

The novel GSTIIIc DNA was isolated from maize (Zea mais) of the Mutinvariety. This DNA codes for the protein GSTIIIc with the amino-acidsequence shown in SEQ ID NO: 2. In plant cells, 2 molecules of theprotein GSTIIIc spontaneously form the dimeric active enzyme (referredto as "GSTIIIc enzyme" hereinafter), which ensures detoxification of theherbicide employed and thus makes the plants resistant to the herbicide.

The GSTIIIc DNA which is preferred according to the invention has thesequence listed in SEQ ID NO: 1.

Likewise preferred according to the invention is the GSTIIIc DNA as iscontained on the vector plasmids pET3a-GSTIIIc and pSS-GSTIIIc describedhereinafter.

The novel GSTIIIc DNA can be in the form of a single strand or in theform of the double strand which additionally contains a strandcomplementary to the particular single strand.

In a preferred embodiment of the present invention, the GSTIIIc DNA hasa promoter which is effective in plants inserted upstream at the 5' end.The usual promoters which are effective in plants can be used for thispurpose. An example which may be mentioned is the promoter of the geneof the small subunit of ribulose-1,5-biphosphate carboxylase (rbsc)(compare EMBO Journal, vol. 5 No. 9, 2063-2071 (1986)). Promoters fromplant viruses are preferably employed, mention being made of the CaMV35S RNA promoter as example. The known construct of the CaMV 35Senhancer and the CaMV 35S promoter which follows in the 5'-3' sequence("CaMV 35S double promoter") is particularly preferably used. Acorresponding preferred construct of the GSTIIIc DNA and the CaMV doublepromoter is present on the vector plasmid pSS-GSTIIIc which is explainedhereinafter. However, it is also possible to use the natural promoterwhich regulates the expression of GSTIIIc DNA in maize, Mutin variety.

The nature of the 3' termination sequence which follows the GSTIIIc DNAin the 5'-3' sequence can be varied extremely widely and is not ofcrucial importance for the present invention. Plant 3'-terminationsequences are preferably employed. It is also possible, for example, touse the natural termination sequence of the GSTIIIc gene from maize,Mutin variety.

Likewise part of the present invention is the novel protein GSTIIIc withthe amino-acid sequence shown in SEQ ID NO: 2 and its likewise noveldimer (GSTIIIc enzyme) which, as already stated above, arisesspontaneously from 2 molecules of the protein GSTIIIc after theirformation in plant cells.

The DNA according to the invention and the protein according to theinvention (where appropriate in the dimeric form) also include in eachcase the DNA sequences and protein sequences derived from this DNA andfrom this protein, respectively. DNA and protein with derived sequencesare intended to mean DNA and protein which still have the essentialfeatures of the GSTIIIc DNA and of the protein GSTIIIc (whereappropriate as GSTIIIc enzyme in the dimeric form), respectively, andwhich therefore have essentially the same effect, that is to say producethe herbicide resistance according to the invention to a sufficientextent in plants. In such derived sequences it is possible forindividual DNAs, codons and/or DNA part-sequences or individual aminoacids or amino-acid part-sequences to be absent (in the case of DNA, forexample, by the use of restriction enzymes) and/or to be replaced byother DNAs, codons and DNA part-sequences or amino acids or amino-acidpart-sequences having essentially the same effect. Modifications ofthese types may be present by reason of the degeneracy of the geneticcode or result from the manipulation of the GSTIIIc DNA or of theprotein GSTIIIc or of the GSTIIIc enzyme. The DNA according to theinvention may also contain DNAs, codons or DNA sequences whichfacilitate its manipulation, for example so-called linkers or whatremain from such linkers after manipulations (for example after cuttingwith restriction enzymes). The GSTIIIc DNA and the protein GSTIIIc orthe GSTIIIc enzyme can be of natural origin or be partly or completelyin synthesized form.

Part of the present invention is also a recombinant prokaryotic oreukaryotic DNA which contains the novel GSTIIIc DNA or a DNA derivedtherefrom as "foreign" or "additional" DNA. Examples which may bementioned are viral DNA, microbial DNA (in particular bacterial DNA,such as DNA from Escherichia coli or Agrobacterium tumefaciens) andplant DNA.

The GSTIIIc DNA according to the invention and the DNA sequences derivedtherefrom, and the recombinant prokaryotic or eukaryotic DNA, and theDNA sequences derived therefrom, may be contained as "foreign" or"additional" DNA in vectors (in particular plasmids, cosmids or phages),in transformed or transgenic microorganisms (preferably bacteria such asEscherichia coli or Agrobacterium tumefaciens) and in transformed ortransgenic plant cells and plants or in their DNA. These vectors,microorganisms, plant cells and plants and their DNA are part of thepresent invention. They can, just like the recombinant prokaryotic andeukaryotic DNA, be obtained by the skilled person with knowledge of thepresent description, in particular SEQ ID NO: 1 and SEQ ID NO: 2, bygenerally known and/or usual processes and methods.

Vectors which may be mentioned as particularly preferred are the vectorplasmids pET3a-GSTIIIc and pSS-GSTIIIc. Both vectors contain the GSTIIIcDNA according to the invention as shown in SEQ ID NO: 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows plasmid pET3a-GSTIIIc (5306 bps)

FIG. 2 shows plasmid pSS-GSTIIIc (1000 bps).

The plasmid pET3a-GSTIIIc contains the GSTIIIc DNA on an Ndel/BamHIfragment. To construct this plasmid, the GSTIIIc DNA shown in SEQ ID NO:1 was cloned (Studier F. W., Moffatt B. A., J. Mol. Biol. 189:113-130;Studier F. W., J. Mol. Biol. 219:37-44 (1991)) into the NdeI and BamHIcleavage sites of the vector pET-3a (Novagen/Madison). This plasmid(5306 bps) is depicted in FIG. 1. The direction of the arrow shows thedirection of the promoter and of the gene, and of the GSTIIIc DNA withthe start codon ATG. "Amp" represents the ampicillin resistance gene.

To construct the plasmid pSS-GSTIIIc, the GSTIIIc DNA was purified asXbal/BamHI fragment from the vector pET3a-GSTIIIc and cloned into theplasmid Bluescript-SKII (Xbal/BamHI-linearized; Stratagene).Subsequently, the GSTIIIc DNA was isolated by SstI/Smal restrictioncleavage from the plasmid Bluescript SKII-GSTIIIc obtained in this wayand and ligated into the vector pRT101 (SstI/SmaI-linearized; Toopfer etal. 1987). The GSTIIIc DNA was then purified as EcoRI/SmaI fragment fromthe vector pRT101-GSTIIIc obtained in this way and cloned into thebinary vector pSS (EcoRI/SmaI-linearized; Voβ et al. 1994). In theresulting vector pSS-GSTIIIc, the coding GSTIIIc DNA is under thecontrol of a duplicated 35S RNA promoter from CaMV. The plasmidpSS-GSTIIIc (10000 bps) is depicted as FIG. 2.

The GSTIIIc DNA according to the invention can if required by isolatedby the skilled person from the said plasmids by generally customaryprocesses and methods.

Transformed or transgenic microorganisms according to the inventionwhich may be mentioned as particularly preferred are the Escherichiacoli strains DS pET3a-GSTIIIc and DS pSS-GSTIIIc and their mutants. Thestrain DS pET3a-GSTIIIc contains the plasmid pET3a-GSTIIIc and thestrain DS pSS-GSTIIIc contains the plasmid pSS-GSTIIIc. Mutantsaccording to the invention are those microorganisms which still containthe features essential for carrying out the invention, that is to say inparticular still contain the plasmids pET3a-GSTIIIc and/or pSS-GSTIIIcor DNA sequences derived therefrom. These strains can be grown bygenerally customary methods. The plamids pET3a-GSTIIIc and pSS-GSTIIIccan likewise be isolated from these microorganisms by generallycustomary methods.

The Escherichia coli strain pET3a-GSTIIIc was deposited at the DeutscheSammlung von Mikroorganismen (DSM), Mascheroder Weg 1b, D-38124Braunschweig, Federal Republic of Germany in compliance with theprovisions of the Budapest Treaty on the international recognition ofthe deposit of microorganisms for the purposes of patent procedure (dateof deposit: 17.01.1995). The strain was given the deposit number DSM9677.

As already explained above, the present invention also includestransgenic plants and plant cells (including protoplasts) and parts ofplants (such as callus, leaves, stems, flowers, parts of flowers, roots,tubors, seeds and other propagation material) from such transgenicplants which contain in their genome the GSTIIIc DNA or DNA sequencesderived therefrom and/or a recombinant prokaryotic or eukaryotic DNAaccording to the invention as "foreign" or "additional" DNA.

The transgenic plants according to the invention also include theprogeny of the transgenic plants obtainable according to the inventionand the results of crossing with other plants and their progeny, as longas these transgenic plants contain the GSTIIIc DNA or DNA sequencesderived therefrom as "foreign" or "additional" DNA.

Preferred transgenic plants are those plants which contain in theirgenome the GSTIIIc DNA as shown in SEQ ID NO: 1 as "foreign" or"additional" DNA.

Particularly preferred transgenic plants are those plants which containin their genomes the construct of the CaMV 35S double promoter and theGSTIIIc DNA shown in SEQ ID NO: 1 as "foreign" or "additional" DNA.

Very particularly preferred transgenic plants are those plants whichcontain in their genome the GSTIIIc DNA shown in SEQ ID NO: 1 and/or theconstruct of the CaMV 35S double promoter and the GSTIIIc DNA shown inSEQ ID NO: 1 as "foreign" DNA. No plants, apart from the maize varietyMutin, which contain a DNA corresponding to GSTIIIc DNA in their genomehave hitherto been disclosed.

In connection with the present invention, "foreign" DNA is intended tomean a DNA which is not naturally present in a particular prokaryotic oreukaryotic (including plant) genome but is taken up in this genome onlythrough human intervention (transformation). "Additional" DNA is a DNAwhich although already present in the particular prokaryotic oreukaryotic genome is taken up in this genome in additional amountthrough human interventions (transformation). The "foreign" or"additional" DNA can be incorporated in one or more copies depending onrequirements and the nature of the particular case.

As has already been explained, the main aim of the present invention isto produce novel plants which have an increased resistance toherbicides, preferably to heteroaryloxyacetamide herbicides.

Thus, the novel transgenic plants according to the invention which arepreferred are those which have in addition to the abovementionedproperties (content of "foreign" or "additional" GSTIIIc DNA) anincreased resistance to herbicides, in particular toheteroaryloxyacetamide herbicides, by comparison with the correspondingnon-transgenic plants. Crops of these transgenic plants can thus betreated with herbicides to control unwanted plants without damaging thecrop plants.

The increased herbicide resistance of the transgenic plant cells andplants according to the invention is important for agriculture andforestry, for the cultivation of ornamental plants, the cultivation ofmedicinal plants and plant breeding.

The present invention thus also relates to a process for the productionof transgenic plant cells (including protoplasts) and plants (includingparts of plants and seeds) with increased resistance to herbicides,which is characterized in that

(a) one or more copies of the GSTIIIc DNA and/or recombinant DNAaccording to the invention, which contain the GSTIIIc DNA, which codefor the protein GSTIIIc, are inserted into the genome of plant cells(including protoplasts) and, where appropriate,

(b) complete transformed plants are regenerated from the transformedplant cells (including protoplasts) and are, where appropriate,propagated and, where appropriate,

(c) the required parts of plants (including seeds) are obtained from thetransgenic plants of the parent generation obtained in this way orfurther generations obtained therefrom.

Process steps (a), (b) and (c) can be carried out in the usual way byknown processes and methods.

Transgenic plant cells (including protoplasts) and plants (includingparts of plants and seeds), which contain the GSTIIIc DNA one or moretimes as "foreign" or "additional" DNA, and those transformed plantcells and plants which are obtainable by the above processes, likewiseform part of the present invention.

Parts of the present invention are also the:

(a) use of the GSTIIIc DNA and/or of the recombinant DNA according tothe invention and/or of the recombinant vectors according to theinvention and/or of the transformed microorganisms according to theinvention for transforming plant cells (including protoplasts) andplants (including parts of plants and seeds), the

(b) use of the transgenic plant cells (including protoplasts) and plants(including parts of plants and seeds) according to the invention forproducing propagation material and producing novel plants and theirpropagation material, the

(c) use of the cDNA contained on the plasmid pET3a-GSTIIIc or its parts,and of the DNA sequences corresponding to the sequence information shownin sequence listing SEQ ID NO: 1 for determining DNA which code forGSTIIIc protein or GSTIIIc enzyme in plants, and (in general) in theproduction of transgenic plant cells (including protoplasts) and plants(including parts of plants and seeds), and the use of the proteinsequence encoded by the GSTIIIc DNA of pET3a-GSTIIIc, and of the proteinshown in SEQ ID NO: 2 in the isolation and detection of GSTIIIc DNA (forexample by the customary antibody technique).

A number of different methods is available for inserting the GSTIIIcDNA, where appropriate as construct with the CaMV35S double promoter, as"foreign" or "additional" DNA into the genetic material of plants orplant cells. The gene transfer can take place by the generally customaryknown methods, the skilled person being able to establish the suitablemethod in each case without difficulty.

The Ti plasmid of Agrobacterium tumefaciens is available as vector whichis particularly favourable and can be widely employed for transferring"foreign" or "additional" DNA into the genomes of dicotyledonous andmonocotyledonous plants. The genetic material which codes for theprotein GSTIIIc is inserted, where appropriate together with regulatoryDNA sequences, into the T DNA of suitable Ti plasmids (for exampleZambryski et al., 1983) and transferred by infecting the plant,infecting parts of plants or plant tissues, such as, for example, leafdiscs, stems, hypocotyles, cotyledons, meristems and tissues derivedtherefrom, such as, for example, secondary embryos and calli, or bycoculture of protoplasts with Agrobacterium tumefaciens.

An alternative is incubation of purified DNA which contains the requiredgene or the required DNA in plant protoplasts (for example Hain et al.,1985; Krens et al., 1982; Paszkowski et al., 1984) in the presence ofpolycations or calcium salts and polyethylene glycol.

DNA uptake can also additionally be favoured by an electric field(electroporation) (for example Fromm et al., 1986).

The DNA can also be introduced in a known manner via plant pollen, by"bombarding" pollen or other parts of plants with physically acceleratedparticles which carry the DNA (compare EP-A 0 270 356).

Regeneration of the plants takes place in a known manner with the aid ofsuitable nutrient media (for example Nagy and Maliga 1976).

In a preferred embodiment of the process according to the invention, theGSTIIIc DNA from the plasmid pET3a-GSTIIIc is cloned into a binaryexpression vector (for example Voβ et al. (1994)). The chimeric geneconstruct is then transferred to Agrobacterium tumefaciens (Koncz andSchell 1986).

Alternatively, the chimeric gene construct on the vector pSS-GSTIIIc istransferred in a customary way by direct gene transfer to plantprotoplasts (for example Hain et al., 1985). It is possible in this casefor the plasmid to be in circular form, but it is preferably in linearform.

When this plasmid is used with reporter gene, kanamycin-resistantprotoplasts are then checked for expression of GSTIIIc.

Transformed (transgenic) plants or plant cells are produced by knownmethods, for example by leaf disc transformation (for example Horsch etal. 1985), by coculture of regenerating plant protoplasts or cellcultures with Agrobacterium tumefaciens (for example Marton et al. 1979,Hain et al. 1985) or by direct DNA transfection. Resulting transformedplants are detected either by selection for the expression of thereporter gene, for example by phosphorylation of kanamycin sulphate invitro (Reiss et al 1984; Schreier et al 1985) or by expression ofnopaline synthase (method of Aerts et al. 1983) or of GSTIIIc byNorthern blot analysis and Western blot analysis. The protein GSTIIIccan also be detected in transformed plants in a known manner in Westernblot analyses using specific antibodies.

Cultivation of the transformed plant cells and regeneration to completeplants takes place by generally customary methods using the nutrientmedia suitable in each case.

Both the transformed plant cells and the transformed plants whichcontain the GSTIIIc DNA according to the invention, and which form partof the present invention, show considerably greater resistance toherbicides, in particular to heteroaryloxyacetamide herbicides.

In connection with the present invention, the term "plants" means bothcomplete plants and parts of plants, such as leaves, seeds, tubors,cuttings etc. "Plant cells" include protoplasts, cell lines, plant callietc. "Propagation material" means plants and plant cells which can beused to propagate the transformed plants and plant cells, and is thuslikewise part of the present invention.

The plants on which increased resistance to herbicides can be conferredby incorporation (transformation) of the GSTIIIc DNA according to theinvention include virtually all plants. There is, of course, aparticular need to produce resistance in crop plants such as forestryplants, for example spruces, firs, douglas firs, pines, larches, beechesand oaks, and plants providing foodstuffs and raw materials, for examplecereals (in particular wheat, rye, barley, oats, millet, rice andmaize), potatoes, leguminosae (such as legumes and, in particular,alfalfa, soya beans), vegetables (especially brassicas and tomatoes),fruit (in particular apples, pears, cherries, grapes, citrus fruits,pineapples and bananas), oil palms, tea, cocoa and coffee plants,tobacco, sisal and cotton, and in medicinal plants such as rauwolfia anddigitalis. Those which may be particularly preferably mentioned arepotatoes, sugar beets, sugar cane, cereals such as wheat and barley andsorghum, and rice. The GSTIIIc DNA according to the invention ispreferably incorporated as "foreign" DNA into the genome of plants.

Preferred herbicides against which increased herbicide resistance can beproduced according to the invention belong to the group ofheteroaryloxyacetamides. Particularly preferred in this connection arethe heteroaryloxyacetamides of the general formula (I)

    Het--O--CH.sub.2 --CO--NR.sup.1 R.sup.2                    (I)

in which

Het represents an optionally substituted heteroaromatic radical with,preferably, 5 ring members, which preferably contains at least onenitrogen atom and one sulphur or oxygen atom, a particularly preferredradical which may be mentioned being the5-trifluoromethyl-1,3,4-thiadiazol-2-yl radical;

R¹ represents optionally substituted alkyl or alkoxy (with, in eachcase, preferably 1-4 carbon atoms); and

R² represents an optionally substituted aryl radical (preferably phenylradical which is preferably substituted by halogen).

Herbicides of this type have already been disclosed (compare, forexample, EP-A-18 497 and the corresponding U.S. Pat. No. 4,645,525,EP-A-94 541 and the corresponding U.S. Pat. No. 4,585,471, and EP-A-348737 and the corresponding U.S. Pat. No. 4,968,342). The herbicidesmentioned in these patent applications and patents are particularlypreferred in connection with the present invention.

Resistance to the herbicide with the chemical name(5-trifluoromethyl-1,3,4-thiadiazol-2-yloxy)acetic acidN-isopropyl-N-(4-fluorophenyl)amide (proposed common name:thiafluamide), which is mentioned in EP-A-348 737 and the correspondingU.S. Pat. No. 4,968,342, is very particularly preferred according to theinvention. This resistance permits the said herbicide to be employedeven in crops which, in the non-resistant form, would be damaged in anunacceptable manner by the herbicide.

The present invention is to be explained in detail by means of thefollowing exemplary embodiments:

I. Isolation of the GSTIIIc DNA from maize

The known processes and methods of molecular biology are used forisolating the GSTIIIc DNA, as are described, for example, in thefollowing handbook: Maniatis T., Fritsch E. F., Sambrook J.: MolecularCloning, A Laboratory Manual; Cold Spring Habor Laboratory, SecondEdition 1989.

To isolate the GSTIIIc DNA, initially the protein is purified frometiolated maize seedlings (Zea mais), Mutin variety (1) and theamino-acid sequence is completely determined by protein sequenceanalysis (2). mRNA is likewise isolated from maize seedlings (3) andtranscribed into cDNA enzymatically by reverse transcriptase (4). ThecDNA obtained in this way is employed as template in the polymerasechain reaction (Mullis K. B., Faloona F. A., (1987) Methods Enzymol.155:335-350) to isolate the GSTIIIc DNA.

1. Isolation of the GSTIIIc protein from maize, variety, Mutin

To purify the GSTIIIc protein, maize seedlings are disintegrated andmixed with 0.2M tris/HCl pH 7.8, 1 mM EDTA (2 ml/g fresh weight). Thesuspension is centrifuged and the protein fraction in the supernatant isobtained by fractional ammonium sulphate precipitation with a saturationof 30% and 70%. The GSTIIIc protein is isolated by chromatography on thefollowing columns with the stated buffer conditions:

A) Sephadex G-100 (dextran-based separating medium), v=500 ml(Pharmacia) Buffer A: 50 mM potassium phosphate pH 7.3

B) DEAE-Sepharose (crosslinked agarose matrix with covalently bondeddiethylaminoethyl group), v=50 ml (Pharmacia) Buffer A: 10 mM potassiumphosphate pH 7.3 Buffer B: 1.0M potassium phosphate pH 7.3 Gradient:0-100% B in 500 ml

C) Glutathione-sulphobromophthalein-agarose, v=20 ml (Sigma) Buffer A:50 mM potassium phosphate pH 7.3 Buffer B: 50 mM potassium phosphate pH8.0, 5 mM glutathione

D) Mono Q HR 5/5 (anion exchange material based on crosslinked agarosewith charged --CH₂ N(CH₃)₃ +groups, particle size of 10±5 μm), v=1 ml(Pharmacia) Buffer A: 20 mM tris/HCl pH 7.5 Buffer B: 20 mM tris/HCl pH8.0, 1.0M NaCl Gradient: 0-25% B in 20 ml

2. Protein sequence analysis of the GSTIIIc protein

The GSTIIIc protein isolated from maize (Mutin variety) is reduced,carboxymethylated and dialysed against 0.2M ammonium bicarbonate (GlazerA. N., Delange R. J., Sigman D. S., (1975) Chemical Modification ofProteins, Elsevier Biomedical Press, Amsterdam). Subsequent cleavage ofthe protein with the endoproteases Asp-N (sequencing grade, BoehringerMannheim) or trypsin (TPCK-treated, Worthington) takes place with aprotein/protease ratio of 1:100 at 23° C. for 16 hours. The cleavagereactions are stopped by adding 0.1% trifluoroacetic acid. Insolublepeptides are removed by centrifugation, and soluble peptides areseparated from one another by reversed phase HPLC under the followingconditions:

    ______________________________________                                        Column:                                                                              Vydac C 18 (silica-based separating gel with aliphatic chains                   (C.sub.18)), 0.46 cm × 25 cm                                     Eluent A: 0.1% trifluoroacetic acid                                           Eluent B: 0.1% trifluoroacetic acid, 90% acetonitrile                         Gradient: 0-60% B in 40 min                                                   Flow rate: 0.25 ml/min                                                      ______________________________________                                    

The amino-acid sequence of the isolated peptides is determined using theApplied Biosystems 470A and 473A Protein Sequenators. The completeprotein sequence of the GSTIIIc protein is depicted in SEQ ID NO: 2.

3. Isolation of poly(A)+mRNA from maize (Mutin variety)

The GSTIIIc DNA, the mRNA and the corresponding cDNA contain nucleotidesequences which can be derived from one another. To isolate the GSTIIIcDNA, initially polyadenylated mRNA is prepared from etiolated maizeseedlings. Isolation takes place using Dynabeads oligo (dT)₂₅ inaccordance with the manufacturer's protocol (Dynal, Oslo/Norway;Jakobsen K. S., Breivold E., (1990) Nucleic Acids Res. 18:3669). Thismethod for purifying poly(A)+RNA is based on the base-pair bindingbetween the poly(A)+residues at the 3' end of mRNA and oligo(dT)residues which are covalently bonded on the surface of magnetic metalbeads (Dynabeads). 0.2 g of plant material are employed per milligram ofDynabeads. The mRNA isolated by this method is employed without furtherpurification steps for the enzymatic synthesis of cDNA.

4. Enzymatic synthesis of cDNA

cDNA is prepared with the aid of the first strand cDNA synthesis kit(Pharmacia P-L Biochemicals Inc.). This method is based on the enzymaticactivity of viral DNA polymerases (reverse transcriptases) whichsynthesize DNA according to an RNA template (Maniatis T., Fritsch E. F.,Sambrook J., (1982) Molecular Cloning: A laboratory manual, Cold SpringHarbor Laboratory).

In accordance with the manufacturer's statements, the reaction iscarried out in the following way:

5 μm of poly(A)+mRNA from maize, Mutin variety were mixed with

1 μm of 0.5M dithioeritrit

1 μm of d(T)₁₆₋₁₈ primer

11 μm of reaction mixture (bulk reaction mix) consisting of:

Murine reverse transcriptase, 135 mM tris/HCl, pH 8.3, 204 mM KCl, 27 mMMgCl₂, 0.24 mg/ml BSA, 5.4 mM DATP, dCTP, dGTP, dTTP.

After a reaction time of one hour at 37° C., the mRNA is removed byalkaline hydrolysis. The synthesized cDNA is precipitated and employedas template for the polymerase chain reaction.

5. Amplification, cloning and sequencing of the GSTIIIc DNA

To isolate the GSTIIIc DNA, the nucleotide sequence coding for GSTIIIcis initially amplified using the polymerase chain reaction (Mullis K.B., Faloona F. A., (1987) Methods Enzymol. 155:335-350). The templateused for the reaction is the cDNA prepared from maize mRNA. For specificenrichment of the GSTIIIc DNA, the primers 1 (forward) shown in SEQ IDNO: 3 and primer 2 (reverse) shown in SEQ ID NO: 4 are employed.

Reaction mixtures with the following composition are prepared:

5 μm of cDNA (200 ng/μl)

1 μm of 50 μM primer 1

1 μm of 50 μM primer 2

4 μl of 250 μM dATP, dCTP, dGTP, dTTP

5 μl of 200 mM tris/HCl, pH 8.8, 100 mM KCl, 60 mM (NH₄)₂ SO₄, 15 mM MgCl₂, 1% Triton X-100

34 μl H₂ O

The polymerase chain reaction takes place in a GeneAmp PCR system 9600thermocycler (Perkin Elmer). 1 μl of Pfu Dna polymerase (Stratagene,2500 U/ml) is added as thermostable polymerase. In total, 35 reactioncycles are carried out with the following temperatures and reactiontimes: 45 sec at 94° C., 45 sec at 58° C., 45 sec at 72° C. Theamplified DNA fragment which contains the nucleotide sequence coding forGSTIIIc is purified by agarose gel electrophoresis and cloned into thevector pET 3a as already described. The complete DNA sequence of GSTIIIcDNA (SEQ ID NO: 1) is determined using the Sequenase DNA sequencing kit(United States Biochemical/Cleveland) (Sanger F., Coulson R., (1975) J.Mol. Biol. 94:441-448).

II. Transformation of rice

Rice (Oryza sativa) can be transformed in accordance with the methodsdescribed in the following references:

Zhang H. M., Yang H., Rech E. L., Golds T. J., Davis A. S., Mulligan B.J., (1988) Transgenic rice plants produced by electroporation-mediatedplasmid uptake into protoplasts. Plant Cell Rep. 7:379-384

Zhang W., Wu R., (1988) Efficient regeneration of transgenic plants fromrice protoplasats and correctly regulated expression of the foreign genein the plant. Theor. Appl.. Gen. 76:835-840

Shimamoto K., Terada R., Izawa T., Fujimoto H., (1989) Fertiletransgenic rice plants regenerated from transformed protoplasts. Nature338:274-276

Datta S. K., Peterhans A., Datta K., Potrykus I., (1990) Geneticallyengineered fertile indica-rice recovered from protoplasts. Biotechnol.6:736-740

Hayashimoto A., Li Z., Murai N., (1990) A polyethylene glycolmediatedtransformation system for production of fertile transgenic rice plants.Plant Physiol. 93:857-863

The method of Hayashimoto et al. (1990) was used without modificationsfor transferring the vector pSS-GSTIIIc into rice.

Kanamycin-resistant transformands were checked for the expression ofGSTIIIc in Northern blot experiments. Formation of the GSTIIIc proteinwas detected by specific antibodies. It was possible to show theenzymatic activity of the protein in the crude extract from transformedrice plants by detecting the enzyme-catalyzed modification of theherbicide (5-trifluoromethyl-1,3,4-thiadiazol-2-yloxy)acetic acidN-isoproypl-N-(4-fluorophenyl)amide.

Transgenic rice plants show resistance to the said herbicide bycomparison with non-transformed controls.

III. Transformation of tobacco

a) Culture of tobacco shoots and isolation of tobacco protopastes:

Nicotiana tabacum (Petit Havanna SR1) is propagated as sterile shootculture on hormone-free LS medium (Linsmaier and Skoog 1965). Atintervals of about 6-8 weeks, shoot sections are transferred to fresh LSmedium. The shoot cultures are kept in a growing room at 24-26° C. with12 h of light (1000-3000 lux).

To isolate leaf protoplasts, about 2 g of leaves (about 3-5 cm long) arecut, using a fresh razor blade, into small pieces (0.5 cm×1 cm). Theleaf material is incubated in 20 ml of enzyme solution consisting of K3medium (Nagy and Maliga 1976), 0.4M sucrose, pH 5.6, 2 % cellulose RIO(Serva), 0.5% Macerozym R10 (Serva) for 14-16 h at room temperature. Theprotoplasts are then separated from cell residues by filtration through0.30 mm and 0.1 mm jet screens. The filtrate is centrifuged at 100×g for10 minutes. During this centrifugation there is flotation of intactprotoplasts, which collect in a band at the upper edge of the enzymesolution. The pellet of cell residues and the enzyme solution areaspirated off with a glass capillary. The prepurified protoplasts aremade up to 10 ml with fresh K3 medium (0.4 m sucrose as osmotic agent)and flotation is repeated. The washing medium is aspirated off and theprotoplasts are diluted to 1-2×10⁵ /ml for culture or followinginfection with agrobacteria (coculture). The protoplast concentration isdetermined in a counting chamber.

b) Construction of the vector pSS-GSTIIIc and transfer intoAgrobacterium tumefaciens

The GSTIIIc DNA and the Shine-Delgarno sequence was cut out asXbal/BamHl fragment from the vector pET3a-GSTIlIc, purified and clonedinto the plasmid Bluescript II Sk +/- (Stratagene, La Jolla, Calif.),referred to as pBS-SKII hereinafter. The Xbal and BamHl cleavage siteswere used. Subsequently, the GSTIIIc DNA was cut out, via the Sstl andSmal cleavage sites, from the vector pBS-SKII-GSTIIIc, isolated andligated in the vector pRT101 (Sstl/Smal-linearized) (Topfer R., MatzeitV., Gronenbom B., Schell J., Steinbiβ H. H., (1987) Nucleic Acids Res.14:5890).

Then the GSTIIIc DNA was purified as EcoRI/Smal fragment from the vectorpRT101-GSTIIIc and cloned into the expression vector pSS(EcoR1/Sma1l-linearized, (Voβ A et al, Molec. Breeding 1:15-26 (1995)).

In place of the said vectors, it is possible to employ any otherexpression vectors and shuttle vectors which have appropriate cleavagesites, the skilled person easily being able to make a suitable choice onthe basis of the above statements. The resulting shuttle vectorpSS-GSTIIIc which contains the GSTIIIc DNA is transferred intoAgrobacterium tumefaciens which contains a functional vir region (Konczand Schell 1986, van Haute et al. 1983).

c) Transformation of regenerating tobacco protoplasts by coculture withAgrobacterium tumefaciens

The method of Marton et al. 1979 with slight modifications is usedhereinafter. The protoplasts are isolated as described and incubated ata density of 1-2×10⁵ /ml in K3 medium (0.4M sucrose, 0.1 mg/ml NAA, 0.2mg of kinetin) at 26° C. in the dark for two days under weak light (500lux) for one to two days.

As soon as the first divisions of the protoplasts occur, 30 μl of anagrobacterium suspension from b) in minimal A (Am) medium (density about10⁹ agrobacteria/ml) are added to 3 ml of regenerating protoplasts.Coculturing is carried out at 20° C. in the dark for 3-4 days. Thetobacco cells are then introduced into 12 ml centrifuge tubes, dilutedto 10 ml with sea water (600 mOsm/kg) and pelleted at 60×g for 10minutes. This washing step is repeated 1-2×more in order to remove mostof the agrobacteria. The cell suspension is cultivated at a density of5×10⁴ /ml in K3 medium (0.3 m sucrose) with 1 mg/l NAA (napthyl-1-aceticacid), 0.2 mg/l kinetin and 500 mg/l of the cephalosporin antibioticcefotaxime. The cell suspension is diluted with fresh K3 medium eachweek, and the osmotic value of the medium is reduced successively by0.05M sucrose (about 60 mOsm/kg) per week. Selection with kanamycin (100mg/l kanamycin sulphate (Sigma), 660 mg/g active Km) is started 2-3weeks after the coculture in agarose bead type culture (Shillito et al.1983). Kanamycin-resistant colonies can be distinguished from thebackground of remaining colonies 3-4 weeks after the start of selection.

d) Direct transformation of tobacco protoplasts with DNA. Calciumnitrate/PEG transformation.

About 10⁶ protoplasts in 180 μl of K3 medium are cautiously mixed with20 μl of aqueous DNA solution which contains 20 μg of plasmidpSS-GSTIIIc in a Petri dish. The plasmid pSS-GSTIIIc can be obtained byknown methods from the plasmids pET3a-GSTIIIc, pRT101, pBS-SKII and pSS.Subsequently, 200 μl of fusion solution (0.1M calcium nitrate, 0.45Mmannitol, 25% polyethylene glycol (PEG 6000), pH 9) are cautiouslyadded. After 15 minutes, 5 ml of washing solution (0.275M calciumnitrate pH 6) are added and, after a further 5 minutes, the protoplastsare transferred into a centrifuge tube and pelleted at 60×g. The pelletis taken up in a small amount of K3 medium and cultivated as describedin the next section. Alternatively, the protoplasts can be transformedas described by Hain et al. 1985.

e) Culture of the protoplasts incubated with DNA and a selection ofkanamycin-resistant calli:

A modified bead type culture technique (Shillito et al. 1983) is usedfor the culture and selection of kanamycin-resistant colonies describedhereinafter. One week after treatment of the protoplasts with DNA(compare d), 3 ml of the cell suspension are mixed with 3 ml of K3medium (0.3M sucrose+hormones; 1.2% (Seaplaque) LMT agarose (low meltingagarose, Marine Colloids) in 5 cm Petri dishes. For this purpose,agarose is autoclaved dry, and after addition of K3 medium, brieflyboiled in a microwave oven. After the agarose has solidified, theagarose discs (beads) are transferred with the embedded tobaccomicrocalli for further culture and selection into 10 cm Petri dishes andto each are added 10 ml of K3 medium (0.3M sucrose, 1 mg/l NAA, 0.2 mg/lkinetin) and 100 mg/l kanamycin sulphate (Sigma). The liquid medium ischanged each week. At the same time, the osmotic value of the medium isreduced stepwise.

The replacement medium (K3+Km) is reduced by 0.05M in sucrose (about 60mOsm) each week.

Scheme for the selection of kanamycin-resistant tobacco colonies afterDNA transformation:

    ______________________________________                                            0.4 M   0.3 M   0.25 M                                                                              0.20 M                                                                              0.15 M                                                                              0.10 M                                                                              Sucrose in                                 liquid                                                                        medium                                                                 A ES    K                                                                      1 2 3 4 5 6 Weeks                                                                   after DNA                                                                     uptake                                                               (K3 medium 1 mg/l NAA, 0.2 mg/l kinetin)                                      ______________________________________                                         A = DNA uptake                                                                E = embedding in agarose                                                      S = selection with kanamycin (100 mg/l kanamycin sulphate)                    K = kanamycinresistant colonies can be clearly distinguished from the         background                                                               

f) Regeneration of kanamycin-resistant plants:

As soon as the kanamycin-resistant colonies have reached a diameter ofabout 0.5 cm, half is placed on regeneration medium (LS medium, 2%sucrose, 0.5 mg/l benzylaminopurine BAP) and kept in a growing room at24° C. with 12 h of light (3000-5000 lux). The other half is propagatedas callus culture on LS medium with 1 mg/l NAA, 0.2 mg/l kinetin, 0.1mg/l BAP and 100 mg/l kanamycin sulphate. When the regenerated shootsare about 1 cm in size, they are cut off and placed on 1/2 LS medium (1%sucrose, 0.8% agar) without growth regulators for rooting. The shootsare rooted on 1/2 MS medium with 100 mg/l kanamycin sulphate and latertransferred into soil.

g) Transformation of leaf discs by Agrobacterium tumefaciens

For transformation of leaf discs (Horsch et al. 1985), leaves about 2-3cm long from sterile shoot cultures are cut into discs of 1 cm diameterand incubated with a suspension of appropriate agrobacteria (about 10⁹/ml) (compare c) in Am medium, see below) for about 5 minutes. Theinfected pieces of leaf are kept on MS medium (see below) withouthormones at about 24° C. for 3-4 days. During this time, agrobacteriumgrows over the pieces of leaf. The pieces of leaf are subsequentlywashed in MS medium (0.5 mg/ml BAP, 0.1 mg/ml NAA) and placed on thesame medium (0.8 % agar) with 500 μg/ml cefotaxime and 100 μg/mlkanamycin sulphate. After two weeks, the medium should be renewed.Transformed kanamycin-resistant shoots are visible after a further 2-3weeks.

Biochemical transformation detection method

Neomycin phosphotransferase (NPT II) enzyme assay:

NPT II activity in plant tissue is detected by in situ phosphorylationof kanamycin, as described by Reiβ et al. (1984) and modified bySchreier et al. (1985), as follows. 50 mg of plant tissue arehomogenized in 50 μl of extraction buffer (10% glycerol, 5%2-mercaptoethanol, 0.1% SDS, 0.025% bromophenol blue, 62.5 mM tris pH6.8) with the addition of glass powder on ice and centrifuged in anEppendorf centrifuge at 4° C. for 10 minutes. 50 μl of the supernatantare loaded onto an unmodified polyacrylamide gel (145×110×1.2 mm;resolving gel: 10% acrylamide, 0.33% bisacrylamide, 0.375M tris pH 8.8,stacking gel: 5% acrylamide, 0.165% bisacrylamide, 0.125M tris pH 6.8)and electrophoresed at 4° C. and 60 V overnight. As soon as thebromophenol blue marker runs out of the gel, the gel is washed twicewith distilled water for 10 min and once with reaction buffer for 30 min(67 mM tris maleate, pH 7.1, 42 mM MgCl₂, 400 mM ammonium chloride). Thegel is placed on a glass plate of the same size and covered with 40 mlof 1% strength agarose in reaction buffer which contains the substrateskanamycin sulphate (20 μg/ml) and 20-200 μCi of ³² P ATP (Amersham). Thesandwich gel is incubated at room temperature for 30 min, and then asheet of P81 phosphocellulose paper (Whatman) is placed on the agarose.On top of this are stacked four layers of 3 MM filter paper (Whatmnan)and a few paper towels. Transfer of in situ phosphorylated radioactivekanamycin phosphate to the P81 paper is stopped after 3-4 h. The P81paper is incubated in a solution of proteinase K and 1% sodium dodecylsulphate (SDS) at 60° C. for 30 min and then washed 3-4 times in 250 mlof 10 mM phosphate buffer pH 7.5 at 80° C., dried and autoradiographed(XAR5 film Kodak) at -70° C. for 1-12 h.

All percentage data in the above examples and those hereinafter relateto percentages by weight unless otherwise indicated.

The presence of GSTIIIc DNA in the plant cells and plants obtained inthe above and examples was confirmed by Southern blot analysis.Expression of the GSTIIIc DNA was detected by Northern blot analysis andWestern blots using specific antibodies.

Some of the media employed in the transformation of plants and plantcells are described below:

Am medium

3.5 g K₂ HPO₄

1.5 g KH₂ PO₄

0.5 g Na₃ citrate

0g MgSO₄ ×7H₂ O

1 g (NH₄)₂ SO₄

2 g glucose

ad 1 1

Medium for sterile tobacco shoot culture

micro-elements 1/2 of the concentration of MS salts

Macro-elements 1/2 of the concentration of MS salts

    ______________________________________                                        Fe EDTA      Murashige and Skoog (MS)                                         Myo-inositol                100    mg/l                                         Sucrose  10 mg/l                                                              Agar  8 mg/l                                                                  Vitamins Ca panthotenate 1 mg/l                                                Biotin 10 mg/l                                                                Nicotinic acid 1 mg/l                                                         Pyridoxine 1 mg/l                                                             Thiamine 1 mg/l                                                            pH 5 .7 before autoclaving                                                    ______________________________________                                    

K3 medium

For culturing Nicotiana tabacum petit Havana SR1, Nicotiana tabacumWisconsin 38, and Nicotiana plumaginifolia protoplasts (Nagy and Maliga,1976)

    ______________________________________                                        Macro-elements NH.sub.4NO.sub.3                                                                            2500   mg/l                                         KNO.sub.3 2500 mg/l                                                           CaCl.sub.2 · 2H.sub.2 O 900 mg/l                                     MgSO.sub.4 · 7H.sub.2 O 250 mg/l                                     NaH.sub.2 PO.sub.4 · 1H.sub.2 O 150 mg/l                             (NH.sub.4).sub.2 SO.sub.4 134 mg/l                                            CaHPO.sub.4 · 1H.sub.2 O 50 mg/l                                    Micro-elements H.sub.3 BO.sub.3 3 mg/l                                         MnSO.sub.4 · 1H.sub.2 O 10 mg/l                                      ZnSO.sub.4 · 4H.sub.2 O 2 mg/l                                       KI 0.75 mg/l                                                                  Na.sub.2 MoO.sub.4 · 2H.sub.2 O 0.25 mg/l                            CuSO.sub.4 · 5H.sub.2 O 0.025 mg/l                                   CoCl.sub.2 · 6H.sub.2 O 0.025 mg/l                                  Fe EDTA Na.sub.2 EDTA 37.2 mg/l                                                FeSO.sub.4 · 7H.sub.2 O 27.8 mg/l                                   Inositol  100 mg/l                                                            Sucrose  137 g/l                                                                                   (= 0.4 M)                                              Xylose                       250    mg/l                                        Vitamins Nicotinic acid 1 mg/l                                                 Pyridoxine 1 mg/l                                                             Thiamine 10 mg/l                                                             Hormones NAA 1.0 mg/l                                                          Kinetin 0.2 mg/l                                                             pH 5.6                                                                        Sterilize filters                                                           ______________________________________                                    

Linsmaier and Skoog medium (Linsmaier and Skoog 1965)

For culturing regenerating protoplasts and for tissue culture of tobaccotumours and callus. Linsmaier and Skoog (LS) medium is Murashige andSkoog medium (Murashige and Skoog, 1962) with the followingmodifications:

Thiamine is weighed in in a higher concentration of 0.4 mg/l in place of0.1 mg/I;

Glycine, pyridoxine and nicotinic acid are absent.

    ______________________________________                                        Macro-elements NH.sub.4 NO.sub.3                                                                           1650   mg/l                                         KNO.sub.3 1900 mg/l                                                           CaCl.sub.2 · 2H.sub.2 O 440 mg/l                                     MgSO.sub.4 · 7H.sub.2 O 370 mg/l                                     KH.sub.2 PO.sub.4 170 mg/l                                                   Micro-elements H.sub.3 BO.sub.3 6.2 mg/l                                       MnSO.sub.4 · 1H.sub.2 O 22.3 mg/l                                    ZnSO.sub.4 · 4H.sub.2 O 8.6 mg/l                                     KI 0.83 mg/l                                                                  Na.sub.2 MoO.sub.4 · 2H.sub.2 O 0.25 mg/l                            CuSO.sub.4 · 5H.sub.2 O 0.025 mg/l                                   CoCl.sub.2 · 6H.sub.2 O 0.025 mg/l                                  Fe EDTA Na.sub.2 EDTA 37.2 mg/l                                                FeSO.sub.4 · 7H.sub.2 O 27.8 mg/l                                   Inositol  100 mg/l                                                            Sucrose  30 g/l                                                               Agar  8 mg/l                                                                  Vitamins Thiamine 0.4 mg/l                                                    Hormones NAA 1 mg/l                                                           Kinetin  0.2 mg/l                                                             pH 5.7                                                                      ______________________________________                                    

The following literature may be cited on the transformation of plantsand plant cells:

Fraley R. T., Rogers S. G., Horsch R. B., Sanders P. R., Flick J. S.,Adams S. P., Bittner M. L., Brand L. A., Fink C. L., Fry J. S., FallupiG. R., Goldberg S. B., Hoffmann N. L., Woo S. C. (1983). Expression ofbacterial genes in plant cells. Proc. Natl. Acad. Sci. U.S.A.80:4803-4807.

Fromm M. E., Taylor L. P., Walbot V. (1986) Stable transformation ofmaize after gene transfer by electroporation. Nature 319: 791-793

Hain, R., Stabel, P., Czernilofsky, A. P., Steinbiβ, H. H.,Herrara-Estrella, L., Schell, J. (1985) Uptake, integration, expressionand genetic transmission of a selectable chimeric gene by plantprotoplasts. Mol. Gen. Genet. 199: 161-168

Hernalsteens J. P., Thia-Tong L., Schell J., Van Montagu M. (1984) AnAgrobacterium-transformed cell culture from the monocot Asparagusofficinalis. EMBO J. 3:3039-3041

Herrera-Estrella L., De Block M., Messens E., Hernaisteens J. P., vanMontagu M., Schell J. (1983) EMBO J. 2: 987-995.

Horsch R. B., Fry J. E. Hoffmann N. L., Eichholtz D., Rogers S. G.,Fraley R. T. (1985) A simple and general method for transferring genesinto plants. Science 277: 1229-1231

Krens F. H., Molendijk L., Wullems G. J., Schilperoort R. A. (1982) Invitro transformation of plant protoplasts with Ti-plasmid DNA. Nature296: 72-74

Koncz C., Schell J. (1986) The promotor of T_(L) -DNA gene 5 controlsthe tissue-specific expression of chimaeric genes carried by a novaltype of Agrobacterium linary vector. Mol. Gen. Genet. (1986) 204:338-396

Linsmaier D. M., Skoog F. (1965) Organic growth factor requirements oftobacco tissue cultures. Physiol. Plant 18: 100-127

Marton L., Wullems G. J., Molendijk L., Schilperoort P. R. (1979) Invitro transformation of cultured cells from Nicotiana tabacum byAgrobacterium tumefaciens. Nature 277: 1229-131

Nagy J. I., Maliga P. (1976) Callus induction and plant regenerationfrom mesophyll protoplasts of Nicotiana sylvestris. Z. Pflanzenphysiol.78: 453-455

Paszkowski J., Shillito R. D., Saul M, Mandak V., Hohn T., Hohn B.,Potrykus I. (1984) Direct gene transfer to plants. EMBO J. 3: 2717-2722

Shillito R. D., Paszkowski J. Potrykus I. (1983) Agarose plating andbead type culture technique enable and stimulate development ofprotoplast-derived colonies in a number of plant species. Pl. Cell. Rep.2: 244-247 Van den Elzen P. J. M., Townsend J., Lee K. Y., Bedbrook J.R.

Van den Elzen P. J. M., Townsend J., Lee K. Y., Bedbrook J. R. (1985) Achimaeric resistance gene as a selectable marker in plant cells. PlantMol. Biol. 5: 299-302.

Velten J., Velten L., Hain R., Schell J. (1984) Isolation of a dualplant promotor fragment from the Ti plasmid of Agrobacteriumtumefaciens. EMBO J. 12: 2723-2730

Van Haute E., Joos H., Maes M., Warren G., Van Montagu M., Schell J.(1983) Intergenic transfer and exchange recombination of restrictionfragments clones in pBR 322: a novel strategy for the reversed geneticsof Ti plasmids of Agrobacterium tumefacines. EMBO J. 2: 411-418.

Zambryski P., Joos H., Genetello C., van Montagu M., Schell J. (1983)Ti-plasmid vector for the introduction of DNA into plant cells withoutaltering their normal regeneration capacity. EMBO J. 12: 2143-2150.

Reiss, B., Sprengel, Will H., and Schaller H. (1984) A new sensitivemethod for qualitative and quantitative assay of neomycinphosphotransferase in crude cell tracts. GENE 1081: 211-217

Schreier P. H., Seftor E. A., Schell J. and Bohnert H. J. (1985) The useof nuclear-encoded sequences to direct the light-regulated synthesis andtransport of a foreign protein into plant chloroplasts. EMBO J. 1: 25-32

The following published patent applications may furthermore be cited:

    ______________________________________                                        EP-A 116 718      EP-A-126 546                                                  EP-A 159 418 EP-A-164 597                                                     EP-A 120 515 EP-A-175 966                                                     EP-A-120 516 WO 84/02913                                                      EP-A-172 112 WO 84/02919                                                      EP-A-140 556 WO 84/02920                                                      EP-A-174 166 WO 83/01176                                                      EP-A-122 791                                                                ______________________________________                                    

The increased resistance of the transformed plants according to theinvention may be illustrated by the following example:

Demonstration of the increased resistance of transformed plants:

To test the increased resistance to herterooxyacetamides with herbicidalactivity, the damage to the plants transformed according to theinvention is determined by comparison with control plants. The testherbicide employed is the compound(5-trifluoromethyl-1,3,4-thiadiazol-2-yloxyacetic acidN-isopropyl-N-(4-fluorophenyl)amide.

Seeds of the F1 generation of the transformed plants are sown on naturalsoil with a 3% humus content in pots (d=11 cm) in a glass house. Theplants are grown at a temperature of 23° C. and a relative humidity of70-80%. The treatment with the abovementioned herbicidal compound,formulated as 70 WP (wettable powder), takes place 24 hours after thesowing of the seeds with concentrations which correspond to anapplication rate of 2000-4000 g of active compound/ha.

Evaluation takes place 20 days after the treatment with herbicide. Thepercentage total damage to transgenic plants is assessed comparing withcorresponding control plants treated with the same concentration ofactive compound.

The transformed tobacco plants into which the GSTIIIc DNA according tothe invention was inserted show a distinctly increased resistance tohigh application rates from of the herbicide (2000-4000 g/ha) comparedwith non-transformed corresponding control plants.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 4                                           - -  - - (2) INFORMATION FOR SEQ ID NO: 1:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 666 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION:1..663                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                           - - ATG GCG CCT CTG AAG CTG TAC GGG ATG CCG CT - #G TCC CCC AAC GTG               - #45                                                                   Met Ala Pro Leu Lys Leu Tyr Gly Met Pro Le - #u Ser Pro Asn Val                 1               5 - #                 10 - #                 15              - - GTG CGC GTG GCC ACC GTG CTC AAC GAG AAG GG - #C CTC GAC TTC GAG               - #90                                                                    Val Arg Val Ala Thr Val Leu Asn Glu Lys Gl - #y Leu Asp Phe Glu                                20 - #                 25 - #                 30              - - ATC GTC CCC GTC GAC CTC ACC ACC GGC GCC CA - #C AAG CAG CCC GAC              13 - #5                                                                   Ile Val Pro Val Asp Leu Thr Thr Gly Ala Hi - #s Lys Gln Pro Asp                                35 - #                 40 - #                 45              - - TTC CTC GCC CTC AAC CCT TTC GGC CAG ATC CC - #G GCT CTC GTC GAC              18 - #0                                                                   Phe Leu Ala Leu Asn Pro Phe Gly Gln Ile Pr - #o Ala Leu Val Asp                                50 - #                 55 - #                 60              - - GGA GAC GAA GTC CTC TTC GAG TCC CGT GCG AT - #C AAC CGG TAC ATC              22 - #5                                                                   Gly Asp Glu Val Leu Phe Glu Ser Arg Ala Il - #e Asn Arg Tyr Ile                                65 - #                 70 - #                 75              - - GCC AGC AAG TAC GCG TCG GAG GGC ACG GAC CT - #G CTC CCC GCG ACG              27 - #0                                                                   Ala Ser Lys Tyr Ala Ser Glu Gly Thr Asp Le - #u Leu Pro Ala Thr                                80 - #                 85 - #                 90              - - GCG TCG GCG GCG AAG CTG GAG GTG TGG CTG GA - #G GTG GAG TCG CAC              31 - #5                                                                   Ala Ser Ala Ala Lys Leu Glu Val Trp Leu Gl - #u Val Glu Ser His                                95 - #                100 - #                105              - - CAC TTC CAC CCG AAC GCG TCG CCG CTG GTG TT - #C CAG CTG CTC GTG              36 - #0                                                                   His Phe His Pro Asn Ala Ser Pro Leu Val Ph - #e Gln Leu Leu Val                               110  - #               115  - #               120              - - AGG CCG CTC CTG GGC GGC GCC CCC GAC GCG GC - #G GTG GTG GAG AAG              40 - #5                                                                   Arg Pro Leu Leu Gly Gly Ala Pro Asp Ala Al - #a Val Val Glu Lys                                125 - #               130  - #               135              - - CAC GCG GAG CAG CTC GCC AAG GTG CTC GAC GT - #G TAC GAG GCG CAC              45 - #0                                                                   His Ala Glu Gln Leu Ala Lys Val Leu Asp Va - #l Tyr Glu Ala His                                140 - #               145  - #               150              - - CTG GCC CGC AAC AAG TAC CTC GCC GGG GAC GA - #G TTC ACG CTC GCC              49 - #5                                                                   Leu Ala Arg Asn Lys Tyr Leu Ala Gly Asp Gl - #u Phe Thr Leu Ala                               155  - #               160  - #               165              - - GAC GCC AAC CAC GCG TCC TAC CTG CTC TAC CT - #C AGC AAG ACC CCC              54 - #0                                                                   Asp Ala Asn His Ala Ser Tyr Leu Leu Tyr Le - #u Ser Lys Thr Pro                               170  - #               175  - #               180              - - AAG GCC GGG CTC GTC GCC GCC CGC CCC CAC GT - #C AAG GCC TGG TGG              58 - #5                                                                   Lys Ala Gly Leu Val Ala Ala Arg Pro His Va - #l Lys Ala Trp Trp                               185  - #               190  - #               195              - - GAG GCC ATC GCC GCC CGC CCC GCG TTC CAG AA - #G ACC GTC GCC GCC              63 - #0                                                                   Glu Ala Ile Ala Ala Arg Pro Ala Phe Gln Ly - #s Thr Val Ala Ala                               200  - #               205  - #               210              - - ATC CCC TTG CCC CCG CCG CCC TCC TCC TCG GC - #T TGA                     - #      666                                                                    Ile Pro Leu Pro Pro Pro Pro Ser Ser Ser Al - #a                                               215  - #               220                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 2:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 221 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                           - - Met Ala Pro Leu Lys Leu Tyr Gly Met Pro Le - #u Ser Pro Asn Val        Val                                                                               1               5 - #                 10 - #                 15             - - Arg Val Ala Thr Val Leu Asn Glu Lys Gly Le - #u Asp Phe Glu Ile Val                   20     - #             25     - #             30                  - - Pro Val Asp Leu Thr Thr Gly Ala His Lys Gl - #n Pro Asp Phe Leu Ala               35         - #         40         - #         45                      - - Leu Asn Pro Phe Gly Gln Ile Pro Ala Leu Va - #l Asp Gly Asp Glu Val           50             - #     55             - #     60                          - - Leu Phe Glu Ser Arg Ala Ile Asn Arg Tyr Il - #e Ala Ser Lys Tyr Ala       65                 - # 70                 - # 75                 - # 80       - - Ser Glu Gly Thr Asp Leu Leu Pro Ala Thr Al - #a Ser Ala Ala Lys Leu                       85 - #                 90 - #                 95              - - Glu Val Trp Leu Glu Val Glu Ser His His Ph - #e His Pro Asn Ala Ser                  100      - #           105      - #           110                  - - Pro Leu Val Phe Gln Leu Leu Val Arg Pro Le - #u Leu Gly Gly Ala Pro              115          - #       120          - #       125                      - - Asp Ala Ala Val Val Glu Lys His Ala Glu Gl - #n Leu Ala Lys Val Leu          130              - #   135              - #   140                          - - Asp Val Tyr Glu Ala His Leu Ala Arg Asn Ly - #s Tyr Leu Ala Gly Asp      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Glu Phe Thr Leu Ala Asp Ala Asn His Ala Se - #r Tyr Leu Leu Tyr        Leu                                                                                             165  - #               170  - #               175             - - Ser Lys Thr Pro Lys Ala Gly Leu Val Ala Al - #a Arg Pro His Val Lys                  180      - #           185      - #           190                  - - Ala Trp Trp Glu Ala Ile Ala Ala Arg Pro Al - #a Phe Gln Lys Thr Val              195          - #       200          - #       205                      - - Ala Ala Ile Pro Leu Pro Pro Pro Pro Ser Se - #r Ser Ala                      210              - #   215              - #   220                          - -  - - (2) INFORMATION FOR SEQ ID NO: 3:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                           - - AGCGCATATG GCGGCTCTGA AGCTGTACGG GAT       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 4:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4:                           - - GACGGATCCT CAAGCCGAGG AGGAGGGCGG         - #                  - #               30                                                                    __________________________________________________________________________

We claim:
 1. An isoluted and purified DNA (deoxyribonucleic acid) whichcodes for the protein glutathione S-transferase IIIc (GSTIIIc) as shownin SEQ ID NO:
 2. 2. DNA according to claim 1, which has the sequenceshown in SEQ ID NO:
 1. 3. DNA according to claim 1, which has thesequence of the GSTIIIc DNA which is present on the vector plasmidpET3a-GSTIIIc.
 4. DNA according to claim 1, which is in the form of aDNA double strand.
 5. DNA according to claim 1, in which a plantpromoter is inserted upstream at the 5' end of the coding sequence. 6.DNA according to claim 5, where the CaMV 35S promoter or the CaMV 35Sdouble promoter consisting of the CaMV 35S enhancer and the CaMV 35Spromoter is used as promoter.
 7. Recombinant prokaryotic or eukaryoticDNA which contains the DNA according to claim
 1. 8. Recombinant DNAwhich is present in plants or plant cells and contains the DNA accordingto claim
 1. 9. Vectors which contain the DNA according to claim
 1. 10.Vectors according to claim 9, which is plasmids pET3a-GSTIIIc. 11.Transformed microorganisms which contain the DNA according to claim 1.12. Escherichia coli strain pET3a-GSTIIIC (according to DSM 9677). 13.Transgenic plants which contain in their genome the DNA according toclaim
 1. 14. Transgenic plants which contain in their genome the DNAaccording to claim
 6. 15. Transgenic plants according to claim 13, whichhave a content of glutathione S-transferase IIIc (GSTIIIc) whereappropriate in the dimeric form.
 16. Transgenic plants according toclaim 11 having an increased resistance to herbicides in comparison withthe corresponding non-transgenic plants.
 17. A process for theproduction of transgenic plants wherein(a) the DNA according to claim 1is inserted into the genome of plant cells and (b) complete transformedplants are generate from the transgenic plant cells and are propagated.18. An isolated and purified protein with an amino-acid sequence shownin SEQ ID NO:
 2. 19. Protein according to claim 18 in the dimeric form.20. Transgenic plants according to claim 16 wherein said herbicide is aheterooxyacetamide herbicide.